Separation of active α1 -acid glycoprotein and utilization in the lipoprotein lipase enzyme system

ABSTRACT

This invention relates to a method of separating the active α 1  -acid glycoprotein fraction, a co-factor in the lipoprotein lipase reaction, from urine of nephrotic animals and humans as follows: 
     (a) concentrate the said urine to about 10-20% by volume; 
     (b) precipitate undesired protein at pH 4 with ammonium sulfate; and 
     (c) recover the α 1  -acid glycoprotein fraction from the supernatant, neutralize with solid NaHCO 3 , and purify by dialysis versus H 2  O and lyophilize. 
     This active α 1  -acid glycoprotein fraction is utilized in effective amounts in nephrotic animals to reverse the defect in triglyceride removal caused by the loss of plasma constituents in urine.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education, and Welfare.

This is a division of application Ser. No. 971,484, filed Dec. 20, 1978,now U.S. Pat. No. 4,178,285.

The present invention has as one target a new method of separating theactive α₁ -acid glycoprotein (α₁ -AG) fraction, which is a co-factor inthe lipoprotein lipase reaction, from nephrotic animal and human urine.This method of separation is as follows:

(a) concentrate the urine to about 10-20% by volume;

(b) precipitate undesired protein at pH 4 with ammonium sulfate;

(c) recover the α₁ -acid glycoprotein from the supernatant, neutralizewith solid NaHCO₃, and purify by dialysis versus H₂ O and lyophilize.

This active α₁ -AG fraction is utilized in effective amounts innephrotic animals to reverse the defect in triglyceride removal causedby the loss of plasma constituents in urine.

It was found that the activity of the α₁ -AG fraction depended also uponthe presence of C-II apolipoprotein which is a protein moiety of plasmalipoproteins which binds the lipid moiety to form the hololipoprotein.The enzymatic fracturers of lipids by lipase is, of course, known andexamples in the art are as follows:

U.S. Pat. No. 3,703,591 Bucolo et al

US. Pat. No. 3,759,793 Stork et al

U.S. Pat. No. 3,862,009 Wahlefeld et al

U.S. Pat. No. 3,898,130 Komatsu

U.S. Pat. No. 3,901,763 Horiuchi et al

U.S. Pat. No. 4,056,442 Huang et al

Felts et al, The Physiologist, Vol. 20, No. 4, August 1977.

Staprans et al, Circulation, 56(4), III-245, 1977.

Staprans et al, Biochem. Biophys. Res. Comm., 79(4):1272-1278 (1977).

The production of an active fraction (active αAG) from the proteinpresent in animal and human urine and blood which affects lipolysis andcan be used for replacement therapy is not known but is suggested andintroduced in the present invention.

Recent experimental work has indicated that the active α₁ -AG fractionis actually a complex in which there is an active component or moietyand an inactive component or moiety.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE shows triglyceride clearance in time with fasted ratsinjected with 50 mg of Intralipid.

BACKGROUND OF THE INVENTION

In the development of this invention there was sought for and found innephrotic patients the different proteins, lipoproteins and co-factorslost from plasma into urine that might have a regulatory effect on theenzyme system (lipoprotein lipase) that is responsible for clearing fatfrom the blood. This component or co-factor was identified as aconstituent of plasma and as the α₁ -acid glycoprotein fraction whichhad a function which was previously unknown.

The problem and its solution in summary is as follows:

Utilizing the active α₁ -AG produced by the above method, it is used asa new co-factor in the lipoprotein lipase reaction. There was isolatedan active form of the compound from nephrotic urine that is effectiveboth in vitro and in vivo. The active α₁ -AG fraction increasedlipolysis 100% in the presence of C-II apolipoprotein in a lipoproteinlipase assay system. Rats with induced nephrotic syndrome showed adecrease in triglyceride clearance. Half time was increased from 15minutes to 43 minutes. The injection of the α₁ -AG fraction restored thelipid clearance to normal. These findings suggest that elevated plasmatriglycerides in human nephrotic patients are the direct result ofexcessive loss of α₁ -AG and/or associated components from plasma intourine and the patients are amenable to replacement therapy.

NEPHROTIC SYNDROME OR KIDNEY DISEASE

A severe excess of lipids in the blood has long been recognized as acommon disorder in patients or experimental animals with the kidneydisease characterized by excess of protein in urine (proteinuria) anddecrease of protein in the blood (hypoproteinemia). It has beenestimated that this type of excess of lipids in the blood is associatedwith an increase in the incidence of ischemic heart disease from 48-85times that of the general population of the same age. The elevatedexcess of triglycerides in the blood is considered to result from aslower clearance of chylomicrons and very low density lipoproteins(VLDL) from the circulation, an increased synthesis of VLDL by theliver, or both.

It has been postulated that urinary loss of apolipoproteins and otherco-factors necessary for the clearance of triglycerides (TG) by thelipoprotein lipase (LPL) enzyme system is believed responsible for adefective clearance of chylomicrons and VLDL. In the present inventionthere has been identified an LPL co-factor in urine which is similar oridentical to plasma α₁ -AG and/or an associated component. Inexperimental nephrotic rats with a TG removal defect, this invention hasshown that the α₁ -AG fraction can restore the rate of TG metabolism tonormal.

THE IDENTIFICATION OF α₁ -ACID GLYCOPROTEIN IN URINE FROM NEPHROTICPATIENTS

A protein common to all urine samples from nephrotic patients wasisolated in pure form by preparative polyacrylamide electrophoresis. Itwas identified as human plasma α₁ -AG by the amino acid compositionwhich was similar to known values [Marshall, J. Biol. Chem.,241:4731-4737 (1966)]. In addition, the antibody against human plasma α₁-AG gave a precipitin band against a purified preparation and notagainst human serum alibumin. Sodium dodecylsulfate (SDS) gelelectrophoresis yielded a molecular weight of 45,000 D which is inagreement with the established values for α₁ -AG of plasma [Li et al, J.Biol. Chem. 243:825-832 (1970)]. The mobility of the urinary protein inbasic polyacrylamide gel electrophoresis [Bamburg, et al, Neurobiology,3:162-175 (1973)] was similar to a commercial preparation of purified α₁-AG (Miles Laboratories, Inc., Kankakee, Ill.).

THE ACTIVATION OF LIPOPROTEIN LIPASE BY THE α₁ -AG FRACTION IN VITRO ANDTHE NECESSITY OF APOLIPOPROTEIN C-II

Table I shows the effect of the α₁ -AG fraction from nephrotic urine onthe activity of LPL in the presence and absence of apolipoprotein C-II.If α₁ -AG was added to the assay in the absence of apolipoprotein C-II,α₁ -AG had no effect on the enzyme activity. However, when α₁ -AG wasadded together with apolipoprotein C-II, LPL enzyme activity increasedover 100%. These results show that the α₁ -AG fraction, in the presenceof apolipoprotein C-II, increases the rate of the enzyme reaction.

                  TABLE I                                                         ______________________________________                                        The Effect of the α.sub.1 -AG fraction on Lipoprotein                   Lipase Activity in the In Vitro Assay                                                                FFA Released                                           Assay Mixture          μmoles/hr                                           ______________________________________                                        LPL (from raw milk)    0.03                                                   LPL + C-II (3μg)    1.14                                                   LPL + C-II (3μg) + α.sub.1 -AG (50μg)                                                    2.52                                                   LPL + α.sub.1 -AG (50 μg)                                                                   0.03                                                   LPL + C-II (3μg) + AT (50μg)*                                                                  1.14                                                   ______________________________________                                         *AT = antitrypsin, a plasma glycoprotein with a molecular weight,             carbohydrate content and charge similar to α.sub.1 -AG.            

SEPARATION OF ACTIVITY FROM THE ACTIVE αAG FRACTION

The "active" component can be separated from the αAG by ion exchangechromatography. The αAG does not adhere to DEAE-cellulose in 0.15 M NaCland all protein is eluted when the column is washed with 0.15 M NaCl.The "activity" can be removed from the cellulose by increasing the saltconcentration of the eluant to 0.8 M NaCl. Thus, by using an anionicexchanger "activity" can be isolated free from αAG.

IDENTIFICATION OF ACTIVE COMPONENT OF THE ACTIVE αAG COMPLEX AS AGLYCOSAMINOGLYCAN (GAG)

Preliminary evidence indicated that the active component was aglycosaminoglycan (GAG) as follows:

(1) The active fraction contained glucuronic acid which is a majorcomponent of all GAGs. Activity and glucoronic acid content coincide bygel filtration of the purified component (free of αAG) when carried outin water or guanidine HCl solution.

(2) Activity separated by DEAE-cellulose did not have UV absorption at280 nm indicating the absence of protein.

(3) Activity was precipitated from the active αAG complex by lysozyme, aprotein which has a selective action in precipitating high molecularweight anionic materials such as GAG (Badin et al., J. Clin. Invest.,34:1317, 1955).

(4) The active component stained with Toluidine Blue, which ischaracteristic of GAGs.

Evidence additionally points to the fact that the active GAG associatedwith α₁ -AG is not heparin:

(1) Molecular weight from gel filtration data (in the presence ofguanidine HCl) for the active component appears to be in the vicinity of60,000 D, which is far higher than expected for heparin (8,000 to 20,000D).

(2) Chromatography on DEAE-Sephadex shows that, unlike heparin, activityis eluted below 1 M NaCl. This is suggestive evidence that the activecomponent may belong to a group of compounds called hyaluronic acids.(Schmidt, Biochim. Biophys. Acta, 63:346, 1962).

EXAMPLE 1 Preparation of the active α₁ -Acid Glycoprotein Fraction

α₁ -AG preparation was based on a method described by Hardwicke et al,Clin. Chim. Acta, 6:503-507 (1961). Urine from patients with nephroticsyndrome was used as a source of the α₁ -AG (orosomucoid) fraction.Urine was concentrated to about 10-20% of the original volume using aMillipore Pellicon membrane. The major portion of the protein wasprecipitated at 50% saturation (36 g/100 ml) with ammonium sulfate at pH4.0. The precipitate was removed by centrifugation and the supernatantcontaining the α₁ -AG fraction was immediately neutralized with solidNaHCO₃. Long exposure of the protein to low pH yielded an inactiveproduct. All procedures were performed in ice. The supernatant was thendialyzed extensively against water in the cold (0°-40°) and concentratedin an Amicon Filtration Cell using a UM20 membrane. Alternatively, thesupernatant was dialyzed against distilled H₂ O and then lyophilized.For final purification of the α₁ -AG, the last traces of contaminatingalbumin and other proteins were removed by Affi-Gel Blue (Bio-RadLaboratories) affinity chromatography. This isolation technique can alsobe applied to plasma, peritoneal fluid and other sources of the α₁ -AGfraction.

Where intended for amino acid analysis, α₁ -AG was isolated in pure formby preparative polyacrylamide gel electrophoresis, with a preparativegel system (Savant Instruments, Inc., Hicksville, N.Y.).

Amino acid analysis was performed according to Spackman et al, Anal.Chem., 30:1190-1206 (1958). Protein was hydrolized in vacuo for 22 hr inthe presence of constant boiling HCl.

Sodium dodecylsulfate gel electrophoresis (SDS) was performed accordingto Weber and Osborn, J. Biol. Chem., 244:4406-4412 (1969).

Apolipoprotein C-II (R-glutamic acid) for lipoprotein lipase activationin the in vitro assay was prepared from VLDL as described by Herbert etal, J. Biol. Chem., 248:4941-4946 (1973). VLDL was isolated fromoutdated plasma by ultracentrifugation at a density of 1.030 anddelipidated by butanol-isopropyl ether. VLDL apoprotein was firstchromatographed on Sephacryl S-200 (Pharmacia) and then onDEAE-cellulose using 6 M urea in the eluting buffers. Purifiedapolipoprotein C-II was dialyzed against 0.15 M NaCl, 0.02 M Tris-HCl,pH 8.0 and used as an activator in the LPL assay.

Lipoprotein lipase was prepared from fresh unpasteurized skim milk usingmethods described by Olivecrona et al, Biochem. Biophys. Res. Commun.,43:524-529 (1971). Final purification was carried out on aheparin-Sepharose column. The eluted active fraction was used as"purified" enzyme in the assay mixture.

EXAMPLE 2 In Vitro Studies of LPL Activity

The assay was based on the procedure described by Felts, et al, Biochem.Biophys. Res. Commun., 66:1467-1475 (1975). A 1.0 ml reaction mixtureconsisted of 0.1 ml 1.3 M Tris-HCL, pH 8.6; 0.1 ml 0.02 M CaCl₂ ; 0.3 mlBSA, 15.0%; 0.1 ml of Intralipid (10%, Vitrum, Stockholm, Sweden); 0.03ml apolipoprotein C-II, 100 μg/ml; 0.10 ml enzyme; 0.27 ml was allowedfor additional variables (α₁ -AG fraction) or water. Duplicate sampleswere incubated for 60 min. at 37° C. and the free fatty acid (FFA)content was determined by titration. A reaction mixture lacking theapolipoprotein C-II activator was incubated under the same conditions asa control blank Enzyme activity was calculated as μmoles FFA releasedper hour.

EXAMPLE 3 Effect of the α₁ -AG fraction on Triglyceride Clearance inNephrotic Rats in Vivo

Experimental nephrosis was induced in rats by injecting puromycinaminonucleoside. Table 2 shows that the puromycin injected ratsdisplayed the common disorders of human nephrotic syndrome: proteinuria,hypoproteinemia, and hypertriglyceridemia.

                  TABLE 2                                                         ______________________________________                                        Plasma and Urine Composition of Control                                       and Nephrotic Rats                                                            Animal    Plasma      Plasma     Urine                                        Group (n) Triglycerides                                                                             Proteins   Proteins                                     ______________________________________                                                  mg/dl       g/dl       mg/24hr                                      Control (4)                                                                             55 ± 9.4*                                                                              7.6 ± 0.24                                                                            trace                                        Nephrotic (5)                                                                           138 ± 39.8                                                                             4.5 ± 0.11                                                                            560 ± 149.6                               ______________________________________                                         *mean ± S.D.                                                          

The FIGURE shows the triglyceride clearance in fasted rats injected with50 mg Intralipid. Nephrotic rats displayed a reduced rate of TGclearance: the initial half time was 43 min. compared to normal ratswith an initial half time of 14 min. However, nephrotic rats which wereinjected with the α₁ -AG fraction cleared TG with an initial half timeof 11 min., which is equivalent to the normal rate. Thus, the injectionof the active α₁ -AG fraction reversed the defective triglycerideclearance mechanism in rats with the nephrotic syndrome. The half timesfor normal and treated nephrotic rats are approximate, sincemeasurements were not taken earlier than 30 min. C-II apolipoproteininjection was not necessary in nephrotic rats since this co-factor isnot limiting in the lipoprotein lipase reaction in vivo.

In vivo experiments with nephrotic rats. Adult (250-300 G) male rats(Long-Evans) were used. In these studies the rats were given BerkeleyDiet A and water ad lib. Experimental nephrotic syndrome was induced bynine daily subcutaneous injections of puromycin aminonucleoside (Sigma)at a dose of 1.67 mg/100 g body weight. Urinary protein was monitoredfor indication of the extent of nephrosis. Two days after the lastinjection, rats were fasted for 18 hr and their plasma triglyceridesdetermined from blood collected from a tail vein. Half of the nephroticrats were injected with 0.5 ml Intralipid (50 mg triglycerides) and 0.5ml α₁ -AG fraction (25 mg). For control purposes, in the remaining halfof the nephrotic rats, α₁ -AG fraction was replaced with 0.15 M NaCl.After the injection of Intralipid with or without the α₁ -AG fraction,blood samples from all rats were collected at 30, 60, and 90 min.intervals and plasma triglycerides were determined. Zero-time valueswere calculated from initial plasma triglycerides plus the amount ofIntralipid injected, taking into account the plasma volume (5% bodyweight).

We claim:
 1. A method of separating and utilizing the active α₁ -acidglycoprotein fraction, which contains a co-factor in the lipoproteinlipase reaction, from nephrotic urine as follows:(a) concentrate thesaid urine to about 10-20% by volume; (b) precipitate undesired proteinat pH 4 with ammonium sulfate; (c) recover the α₁ -acid glycoproteinfraction from the supernatant, neutralize with solid NaHCO₃, and purifyby dialysis versus H₂ 0 and lyophilize;wherein the utilization of activeα₁ -acid glycoprotein containing glycosaminoglycan is made in effectiveamounts in nephrotic animals to reverse the effect of the loss of plasmaconstituents in urine.
 2. A method of separating and utilizing theactive α₁ -acid glycoprotein fraction, which contains a co-factor in thelipoprotein lipase reaction, from nephrotic urine as follows:(a)concentrate the said urine to about 10-20% by volume; (b) precipitateundesired protein at pH 4 with ammonium sulfate; (c) recover the α₁-acid glycoprotein fraction from the supernatant, neutralize with solidNaHCO₃, and purify by dialysis versus H₂ O and lyophilize;wherein theutilization of active α₁ -acid glycoprotein containing glycosaminoglycanis made in effective amounts in nephrotic rats to reduce the increasedamount of triglyceride in the blood.